The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
Ansamycins are a class of naturally occurring, synthetic, and semi-synthetic formulas characterized by aliphatic rings of various length and constitution bridging opposite ends of aromatic ring formulas and their reduced equivalents. Subsumed within this class is the sub-class, benzoquinone ansamycins, which possess a benzoquinone as the aromatic ring formula U.S. Pat. Nos. 3,595,955, 4,261,989, 5,387,584, and 5,932,566, and International Applications PCT/US92/10189 (WO 93/14215) and PCT/IB94/00160 (WO 95/01342) describe the isolation, characterization, preparation and/or use of many ansamycins, including the well-known benzoquinone ansamycin, geldanamycin, and its hydrogenated equivalents. See also Progress in the Chemistry of Organic Natural Products, Chemistry of the Ansamycin Antibiotics, 33 1976, p. 278.
Geldanamycin, as first isolated from the microorganism Streptomyces hygroscopicus, was originally identified as a potent inhibitor of certain kinases, and was later shown to act by stimulating kinase degradation, specifically by targeting “molecular chaperones”, e.g., heat shock protein 90s (HSP90s). Subsequently, various other ansamyins have demonstrated more or less such activity, with 17 allyl amino geldanamycin (17-AAG) being among the most promising and the subject of intensive clinical studies currently being conducted by the National Cancer Institute (NCI) for their use as potential anti-cancer agents. See, e.g., Federal Register, 66(129): 35443-35444; Erlichman et al., Proc. AACR (2001), 42, abstract 4474.
HSP90s are ubiquitous chaperone proteins that are highly conserved in nature and that are thought to be involved in folding, activation and assembly of a wide range of proteins, including key proteins involved in signal transduction, cell cycle control and transcriptional regulation. NCBI accession #'s P07900 and XM 004515 (human α and β HSP90, respectively), P11499 (mouse), AAB2369 (rat), P46633 (chinese hamster), JC1468 (chicken), AAF69019 (flesh fly), AAC21566 (zebrafish), AAD30275 (salmon), O02075 (pig), NP 015084 (yeast), and CAC29071 (frog) are illustrative of HSP90s. Grp94 and Trap-1 are related molecules that may exhibit a similar effect when contacted with HSP90 inhibitors. Researchers have reported that HSP90s are associated with important signaling proteins, such as steroid hormone receptors and protein kinases, including, e.g., Raf-1, EGFR, v-Src family kinases, Cdk4, and ErbB-2 (Buchner J., 1999, TIBS, 24: 136-141; Stepanova, L. et al., 1996, Genes Dev. 10: 1491-502; Dai, K. et al., 1996, J. Biol. Chem. 271: 22030-4). Studies further indicate that certain co-chaperones, e.g., Hsp70, p60/Hop/Stil, Hip, Bag1, HSP40/Hdj2/Hsj1, immunophilins, p23, and p50, may assist HSP90 function (see, e.g., Caplan, A., 1999, Trends in Cell Biol., 9: 262-68).
Ansamycins are thought to exert their anti-cancerous effects by tight binding of the N-terminus pocket of HSP90s (Stebbins, C. et al., 1997, Cell, 89: 239-250). This pocket is highly conserved and has weak homology to the ATP-binding site of DNA gyrase (Stebbins, C. et al., supra; Grenert, J. P. et al., 1997, J. Biol. Chem., 272: 23843-50). Further, ATP and ADP have both been shown to bind this pocket with low affinity and to have weak ATPase activity (Proromou, C. et al., 1997, Cell, 90: 65-75; Panaretou, B. et al., 1998, EMBO J., 17: 4829-36). In vitro and in vivo studies have demonstrated that occupancy of this N-terminal pocket by ansamycins and other HSP90 inhibitors alters HSP90 function and inhibits protein folding. At high concentrations, ansamycins and other HSP90 inhibitors have been shown to prevent binding of protein substrates to HSP90 (Scheibel, T., H. et al., 1999, Proc. Natl. Acad. Sci. USA 96: 1297-302; Schulte, T. W. et al., 1995, J. Biol. Chem. 270: 24585-8; Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci. USA 91: 8324-8328). Ansamycins have also been demonstrated to inhibit the ATP-dependent release of chaperone-associated protein substrates (Schneider, C., L. et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 14536-41; Sepp-Lorenzino et al., 1995, J. Biol. Chem. 270: 16580-16587). In either event, the substrates are degraded by a ubiquitin-dependent process in the proteasome (Schneider, C., L., supra; Sepp-Lorenzino, L., et al., 1995, J. Biol. Chem., 270: 16580-16587; Whitesell, L. et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 8324-8328).
This substrate destabilization occurs in tumor and non-transformed cells alike and has been shown to be especially effective on a subset of signaling regulators, e.g., Raf (Schulte, T. W. et al., 1997, Biochem. Biophys. Res. Commun. 239: 655-9; Schulte, T. W., et al., 1995, J. Biol. Chem. 270: 24585-8), nuclear steroid receptors (Segnitz, B., and U. Gehring. 1997, J. Biol. Chem. 272: 18694-18701; Smith, D. F. et al., 1995, Mol. Cell. Biol. 15: 6804-12), v-src (Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci. USA 91: 8324-8328) and certain transmembrane tyrosine kinases (Sepp-Lorenzino, L. et al., 1995, J. Biol. Chem. 270: 16580-16587) such as EGF receptor (EGFR) and Her2/Neu (Hartmann, F., et al., 1997, Int. J. Cancer 70: 221-9; Miller, P. et al., 1994, Cancer Res. 54: 2724-2730; Minmaugh, E. G., et al., 1996, J. Biol. Chem. 271: 22796-801; Schnur, R. et al., 1995, J. Med. Chem. 38: 3806-3812), CDK4, and mutant p53. Erlichman et al., Proc. AACR (2001), 42, abstract 4474. The ansamycin-induced loss of these proteins leads to the selective disruption of certain regulatory pathways and results in growth arrest at specific phases of the cell cycle (Muise-Heimericks, R. C. et al., 1998, J. Biol. Chem. 273: 29864-72), and apoptsosis, and/or differentiation of cells so treated (Vasilevskaya, A. et al., 1999, Cancer Res., 59: 3935-40).
Recently, Nicchitta et al., WO 01/72779 (PCT/US01/09512), demonstrated that HSP90 can assume a different conformation upon heat shock and/or binding by the fluorophore bis-ANS. Specifically, Nicchitta et al. demonstrated that this induced conformation exhibits a higher affinity for certain HSP90 ligands than for a different form of HSP90 that predominates in normal cells. Commonly-owned application PCT/US02/39993carries this discovery even further by demonstrating the utility and uses of cancer cell lystates as excellent sources of high affinity HSP90.
In addition to anti-cancer and antitumorgenic activity, HSP90 inhibitors have also been implicated in a wide variety of other utilities, including use as anti-inflammation agents, anti-infectious disease agents, agents for treating autoimmunity, agents for treating stroke, ischemia, cardiac disorders and agents useful in promoting nerve regeneration (See, e.g., Rosen et al., WO 02/09696 (PCT/US01/23640); Degranco et al., WO 99/51223 (PCT/US99/07242); Gold, U.S. Pat. No. 6,210,974 B1; DeFranco et al., U.S. Pat. No. 6,174,875). Overlapping somewhat with the above, there are reports in the literature that fibrogenetic disorders including but not limited to scleroderma, polymyositis, systemic lupus, rheumatoid arthritis, liver cirrhosis, keloid formation, interstitial nephritis, and pulmonary fibrosis also may be treatable. (Strehlow, WO 02/02123; PCT/US01/20578). Still further HSP90 modulation, modulators, and uses thereof are reported in PCT/US02/35938, PCT/US02/16287, PCT/US02/06518, PCT/US98/09805, PCT/US00/09512, PCT/US01/09512, PCT/US01/23640, PCT/US01/46303, PCT/US01/46304, PCT/US02/06518, PCT/US02/29715, PCT/US02/35069, PCT/US02/35938, PCT/US02/39993, 60/293,246, 60/371,668, 60/331,893, 60/335,391, 06/128,593, 60/337,919, 60/340,762, 60/355,275, 60,367,055 and 60/359,484.
However, at present the various known methods of producing ansamycins exhibit one or more of low yield, low purity, low solubility, chemical instability (in vivo and/or in vitro), poor pharmaceutical properties (short t1/2, metabolic instability), environmental toxicity associated with the use of halogenated organic solvents, and additional attendant costs in terms of time, expense, waste disposal, and health risks to those taking the drugs so made. Commonly-owned applications, Ser. Nos. 60/272,251, filed Mar. 1, 2001, and entitled Methods for Treating Genetically Defined Proliferative Disorders with HSP90; Ser. Nos. 60/326,639 and 60/331,893, filed respectively Sep. 24, 2001 and Nov. 21, 2001, and both entitled Chemical Process for Preparing 17-Allyl Amino Geldanamycin (17-AAG) and other Ansamycins and Ansamycin Derivatives; and a provisional application filed Dec. 2, 2001, and entitled Assay for Determining HSP90 Binding Activity, describe some of these problems and how they may be addressed.
It is an object of the present invention to ameliorate one or more of the problems associated with traditional ansamycin preparations and use, e.g. low metabolic stability, low bioavailability, low water solubility and/or formulation difficulty. Another object is to provide new ansamycin compounds, preferably of improved potency and pharmacokinetic properties.